Piscine myocarditis virus-like particles and methods of use thereof

ABSTRACT

The present invention relates to a piscine myocarditis virus (PMCV) virus-like particle (VLP) which consists only of PMCV ORF1 protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to GB 1701573.6, filed Jan. 31, 2017,all contents of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to piscine myocarditis virus (PMCV)virus-like particles (VLP), and their use, for example as a medicamentto treat or prevent cardiomyopathy syndrome (CMS) in fish.

BACKGROUND

CMS is an inflammatory heart disease, primarily affecting farmedAtlantic salmon, Salmo salar L. The disease was first detected in Norwayin 1985 and has since been diagnosed in both farmed and wild Atlanticsalmon.

CMS is most commonly diagnosed during the late sea-water phase in farmedsalmon and is a disease causing considerable losses for the salmonindustry. The clinical features of CMS vary from acute death withoutprior clinical signs to elevated mortality with nonspecific signs suchas impaired or abnormal swimming behaviour. Diagnosis of CMS is based ondetection of characteristic inflammation and degeneration of spongymyocardium in the atrium and ventricle during histopathologicalexamination.

The causative agent of CMS has been identified as piscine myocarditisvirus (PMCV) and the isolation and characterisation of the virus isdescribed in WO 2011/131600.

Methods of controlling the virus are urgently required but despiteintensive research, it has not been possible to efficiently culturePMCV, or consequently to produce vaccines based on the attenuated orinactivated virus. Current approaches to vaccinating against CMS are,therefore, based on recombinant PMCV proteins, produced by recombinantexpression systems in which no virus is used. Such vaccines, however, donot present the viral proteins in the natural structural context.

SUMMARY

In a first aspect, the present invention provides a recombinant piscinemyocarditis virus (PMCV) virus-like particle (VLP) which consists onlyof PMCV ORF1 protein.

Preferably, the recombinant PMCV VLP consists only of PMCV ORF1 proteinwhich has at least 70% sequence identity to the sequence set out in SEQID No: 2. In one embodiment the recombinant PMCV VLP consists only ofPMCV ORF1 protein having between 70%-90% sequence identity to thesequence set out in SEQ ID No: 2. In one embodiment, the recombinantPMCV VLP consists only of PMCV ORF1 protein having at least 90% sequenceidentity to the sequence set out in SEQ ID No: 2.

Preferably, the recombinant PMCV VLP consists only of PMCV ORF1 proteinwhich has a sequence which differs from the PMCV ORF1 protein sequenceof SEQ ID No: 2 by no more than 80 amino acid residues. For example, thePMCV VLP may consist only of PMCV ORF1 protein which has a sequencewhich differs from the PMCV ORF1 protein sequence of SEQ ID No: 2 by nomore than 15 amino acid residues.

In one or more embodiments the recombinant PMCV VLP may consist only ofPMCV ORF1 protein having the sequence, or a fragment of the sequence,set out in SEQ ID No: 2.

In a second aspect, the present invention provides a method for theproduction of a recombinant PMCV VLP which consists only of PMCV ORF1protein. The method comprises:

i) infecting an insect cell with a baculovirus expression vectorencoding the PMCV ORF1 protein of the present invention; and,

ii) incubating the infected cell to allow the expression of PMCV ORF1proteins for the production of a PMCV VLP.

Preferably, step i) infecting an insect cell with a baculovirusexpression vector comprises incubating the cells at 27° C. for 24 hours.

Preferably, step ii) suitably incubating the infected cell comprisesincubating the cell at a temperature in the range of 12-20° C. for aperiod of 2-10 days. For example, the cell may be incubated at atemperature in the range of 15-17° C. and/or for a period of 2-4 days.

In a third aspect, the invention provides a recombinant PMCV VLP whichis obtained or obtainable by a method of the second aspect.

In a fourth aspect, the invention provides a recombinant PMCV VLP of thefirst aspect or the third aspect for use as a medicament. For example,the recombinant PMCV VLP may be suitable for use in treating orpreventing cardiomyopathy syndrome (CMS) infections of fish.

In a fifth aspect, the present invention provides a vaccine comprising arecombinant PMCV VLP of the first aspect or third aspect for use intreating or preventing cardiomyopathy syndrome (CMS) infections in fish.In one embodiment the vaccine of the invention may further comprise atleast one other antigen of a fish-pathogenic microorganism or afish-pathogenic virus. In one embodiment the vaccine of the inventionmay comprise an adjuvant.

In a sixth aspect, the present invention provides a baculovirusexpression vector comprising genetic information encoding the expressionof PMCV ORF1 protein as defined in relation to the first aspect. Forexample, the baculovirus expression vector may comprise geneticinformation encoding only the expression of PMCV ORF1 protein as definedin relation to the first aspect.

In a seventh aspect, the present invention provides an insect cellcomprising a baculovirus expression vector of the fourth aspect.

In an eighth aspect, the present invention provides the use of abaculovirus based expression system for the production of the PMCV VLPof the first aspect or third aspect.

In a ninth aspect, the invention provides a PMCV VLP substantially asdescribed herein.

FIGURES

FIG. 1 shows the nucleotide sequence of the ORF1 of a specific isolateof PMCV, which corresponds to the sequence set out in SEQ ID No: 1.

FIG. 2 shows the amino acid sequence of the ORF1 of a specific isolateof PMCV, which corresponds to the sequence set out in SEQ ID No: 2, andwhich is encoded by the nucleotide sequence of SEQ ID No: 1 and FIG. 1.

FIG. 3 is an electron micrograph showing PMCV virus-like particlesproduced in Tni cells expressing recombinant PMCV ORF1 protein only, asdescribed in Example 6.

FIG. 4 is an electron micrograph showing PMCV virus-like particlesproduced in Tni cells expressing recombinant PMCV ORF1 and ORF3 proteinsin combination, as described in Example 6.

DETAILED DESCRIPTION

PMCV is a double stranded RNA virus, with three open reading frames(ORF1, ORF2, and ORF3). PMCV has been classified in the familyTotiviridae based on a low level of sequence identity between the PMCVORF2 protein and an RNA-dependent RNA polymerase (RdRp) fromTotiviridae. The other PMCV proteins, ORF1 and ORF3, however, have nosignificant homology to any other known proteins. The function of theseproteins cannot be determined by homology to proteins from otherTotiviridae due to the insufficient degree of sequence identity. Variousother lines of evidence, however, have indicated that both ORF1 and ORF3are coat proteins required for formation of the PMCV capsid. Forexample, in WO 2011/131600 both ORF1 and ORF3 are described as coatproteins. WO 2011/131600 also includes examples showing that antibodiesthat recognise the PMCV ORF1 or ORF3 proteins are capable of binding tointact CMS virus particles, indicating that at least a part of both theORF1 and ORF3 proteins must be present on the surface of the virus.

Haugland et al (J Virol. 85 (11): 5275-5286), describe that PMCV is inmany ways more similar to the putative totivirus Infectious MyonecrosisVirus (IMNV) of shrimp than other members of the family Totiviridae. Forexample, almost all other totiviruses asymptomatically and persistentlyinfect fungi and protozoans and are transmitted to new host cells onlyduring cell division, sporogenesis, and cell fusion. In contrast, bothPMCV and IMNV can be transmitted extracellularly through the release ofvirus particles from infected cells, and both viruses cause myonecrosisin the host. The IMNV virions have fibre-like protrusions which areabsent in other members of the family Totiviridae. The surfaceprotrusions of IMNV are of a similar molecular weight and organisationto the PMCV ORF3 protein and in view of this, and the similarities inthe modes of viral transmission, it is suggested that the ORF3 proteinis a PMCV surface protein (Haugland et al).

The present application, however, is based on the surprising discoverythat ORF3 is not required for the formation of the PMCV capsid and thatPMCV virus-like particles can be made consisting only of ORF1 protein.

Virus-like particles are produced from viral coat proteins andstructurally resemble the natural virion, and are, therefore, highlyimmunogenic, but contain no genetic material and are not infectious. Tothe inventors' knowledge, synthetic virus-like particles of any membersof the family Totiviridae have not previously been produced, and for thefirst time, PMCV VLP, and methods of producing PMCV VLP, are nowdescribed in the present disclosure.

The genetic sequence of the PMCV ORF1 of the isolated PMCV described inWO 2011/131600 is given in SEQ ID No: 1. The corresponding proteinsequence of the PMCV ORF1 is given in SEQ ID No. 2.

The protein sequence given in SEQ ID No. 2 is the protein sequence ofone particular PMCV isolate, and as such is merely an example of aparticular PMCV ORF1 protein. Due to genetic variation that inevitablyexists between individual PMCV virus particles within the population asa whole, other PMCV ORF1 protein sequences also exist. For the purposesof the present invention, a PMCV ORF1 protein is considered to be anyPMCV ORF1 protein encoded in the genome of any PMCV virus, which istranscribed from an open reading frame corresponding to the open readingframe designated for the purposes of the present invention (andconventionally in the PMCV field) as ORF1.

Thus, in some embodiments, the PMCV VLP may consist only of PMCV ORF1protein having a protein sequence that is identical to, or substantiallyidentical to, the sequence, or a fragment of the sequence, set out inSEQ ID No: 2. For the avoidance of doubt, the terms “substantiallyidentical”, “substantially similar” or “substantially the same” denotesa sufficiently high degree of similarity between two numeric values(such as one associated with the ORF1 protein sequence of a given PMCVand the other associated with the ORF1 protein sequence set out in SEQID No: 2) such that the skilled person would consider the differencebetween the two values to be of little or no biological and/orstatistical significance within the context of the biologicalcharacteristic measured by said values (such as sequence identity).Unless otherwise stated, the difference between the two values is lessthan about 20%, 10%, 7%, 5%, or preferably less than about 3%, 2%, 1.5%,1%, or 0.5% of the reference value.

However, the genome sequences of individual PMCV viruses in thepopulation as a whole are not identical and, as a result, the sequencesof PMCV ORF1 proteins within the population may differ. The skilledperson would understand that a particular PMCV ORF1 protein may notnecessarily have a protein sequence that is identical to, orsubstantially identical to, the sequence given in SEQ ID No: 2. Thus, insome embodiments, the PMCV VLP may consist only of a protein or fragmenthaving a sequence which has at least 70% sequence identity to the PMCVORF1 protein sequence set out in SEQ ID No: 2. For example, the PMCV VLPmay consist only of a protein or fragment having a sequence which has atleast 75%, 80%, 85%, 90%, 95%, or 97% sequence identity to the PMCV ORF1protein sequence set out in SEQ ID No: 2. In preferred embodiments, thePMCV VLP consists of a protein or fragment having a sequence which hasat least 97%, 97.5%, 98%, 98.5%, 99%, 99.5% sequence identity to thePMCV ORF1 protein sequence set out in SEQ ID No: 2.

The PMCV ORF1 protein of SEQ ID No: 2 has 861 amino acid residues intotal. In some embodiments, the PMCV VLP of the invention consists onlyof a protein or fragment having a sequence which differs from the PMCVORF1 protein sequence of SEQ ID No: 2 by no more than 80, 60, 50, 40,30, or 20 amino acid residues. Preferably, the PMCV ORF1 proteinsequence of the invention differs from that of SEQ ID No: 2 by no morethan 15, 12, 10, 8, 7, 6, or 5 amino acid residues. Even morepreferably, PMCV ORF1 protein sequence of the invention differs from theprotein sequence of SEQ ID No: 2 by only 4, 3, 2, or 1 amino acid. Insome embodiments the PMCV ORF1 protein sequence of the invention differsfrom the protein sequence of SEQ ID No: 2 by amino acid substitutions inone or more of the following specific positions (corresponding to theresidue numbers of the sequence set out in SEQ ID No: 2): 37, 40, 57,248, 300, 401, 442, 484, 486, 490, 587, 645, 655, 773, and/or 774. Insome embodiments the PMCV ORF1 protein sequence of the invention differsfrom the protein sequence of SEQ ID No: 2 by one or more of thefollowing specific amino acid substitutions: A to V at position 37, A toV at position 40, G to S at position 57, S to N at position 248, D to Gat position 300, A to T at position 401, M to T at position 442, A to Gat position 484, A to P at position 486, P to L at position 490, M to Tat position 587, Y to H at position 645, H to R at position 655, T to Aat position 773, and/or A to V at position 774.

The PMCV ORF1 nucleotide sequence of SEQ ID No: 1 has 2586 nucleotidesin total. Due to the redundancy in the genetic code it would be possibleto alter at least 800 nucleotides of the sequence of SEQ ID No: 1without changing the identity of a single amino acid residue of theencoded amino acid sequence from that set out in SEQ ID No: 2. Thus, insome embodiments, the PMCV VLP of the invention consists only of aprotein or fragment encoded by a sequence which differs from the PMCVORF1 nucleotide sequence of SEQ ID No: 1 by no more than 1200, 1100,1000, 950, 900, or 850 nucleotides. In some embodiments, the PMCV VLPmay consist only of a protein or fragment encoded by a nucleotidesequence which has at least 70% sequence identity to the PMCV ORF1nucleotide sequence set out in SEQ ID No: 1. For example, the PMCV VLPmay consist only of a protein or fragment encoded by a nucleotidesequence which has at least 75%, 80%, 85%, 90%, 95%, 97%, or 99%sequence identity to the PMCV ORF1 nucleotide sequence set out in SEQ IDNo: 1.

For the avoidance of doubt, the terms “% sequence identity”, “% sequencesimilarity”, “% identical”, “% identity”, “% homology”, and similarterms, are to be understood to refer to the percentage of nucleotides oramino acid residues that two or more sequences or fragments, whenoptimally aligned, contain that are the same, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. The skilled person willacknowledge that various means for comparing sequences are available.For example, one non-limiting example of a computer sequence alignmentprogram is the Basic Local Alignment Search Tool (BLAST). Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. The % amino acid (ornucleotide) sequence identity of a given amino acid (or nucleotide)sequence A to, with, or against a given amino acid (or nucleotide)sequence B (which can alternatively be phrased as a given amino acid (ornucleotide) sequence A that has or comprises a certain % amino acid (ornucleotide) sequence identity to, with, or against a given amino acid(or nucleotide) sequence B) is calculated as [100 times the fractionX/Y] where X is the number of amino acid (or nucleotide) residues scoredas identical matches in the alignment of A and B, and where Y is thetotal number of amino acid (or nucleotide) residues in B. It will beappreciated that where the length of amino acid (or nucleotide) sequenceA is not equal to the length of amino acid (or nucleotide) sequence B,the % amino acid (or nucleotide) sequence identity of A to B will notequal the % amino acid (or nucleotide) sequence identity of B to A. Aspecified percentage of nucleotides or amino acid residues can bereferred to as having, for example, 70%, 80%, 85%, 90%, 95%, 99%sequence identity or homology over a specified region when compared andaligned for maximum correspondence. Unless otherwise stated, thedetermination of sequence identity/homology is based on a comparison ofthe entire sequences, and not on selected portions.

In some embodiments, the PMCV VLP may consist only of PMCV ORF1 proteinwhich is a fragment or variant of the protein sequence set out in SEQ IDNo: 2, and/or which is encoded by a fragment or variant of thenucleotide sequence of SEQ ID No: 1. The term “variant” used in respectof the nucleic acid sequences and proteins according to the presentinvention is to be understood to encompass nucleic acid sequences andproteins that only differ from the reference sequences by way of aminoacid or nucleotide additions, deletions, or alterations that have littleor no effect on the functional activity of the claimed sequences. Theskilled person will acknowledge that modifications of a protein sequenceor protein coding nucleotide sequence may be introduced which do notalter, or do not significantly alter, the structure or properties of thefolded protein. For example, the substitution of a nucleotide in atriplet codon may not alter the identity of the encoded amino acid.Similarly, alterations of the nucleic acid sequence resulting inmodifications of the amino acid sequence of the correspondingrecombinant protein may have little, if any, effect on the protein'sability to form VLP if the alteration does not have any impact on theresulting three dimensional protein structure. For example, a codon fora hydrophobic amino acid may be substituted by a codon encoding anotherhydrophobic residue. Similarly, changes which result in substitution ofone negatively or positively charged residue for another can also beexpected to produce little or no alteration in the resulting foldedprotein's tertiary structure or function. Therefore, references to“variants” of nucleic acid or protein sequences of the invention will beunderstood to encompass all such modifications which result in theproduction of a biologically equivalent protein.

The PMCV ORF1 protein of the invention must be functional in the sensethat the PMCV ORF1 protein must be capable of forming part of a PMCVVLP. Regardless of the degree of sequence identity that a given proteinhas to the sequence set out in SEQ ID No: 2, the protein cannot beconsidered a PMCV ORF1 protein for the purposes of the invention if itcannot be used in the formation of a PMCV VLP. This clearly sets afunctional limitation on the extent to which a PMCV ORF1 protein of theVLP of the invention may differ from the protein of SEQ ID No: 2; PMCVORF1 proteins that are fragments or variants of the protein of SEQ IDNo: 2 which are not capable of forming part of a PMCV VLP are notconsidered to be PMCV ORF1 proteins for the purposes of the presentinvention.

The PMCV VLP of the invention consists only of PMCV ORF1 protein. Forthe avoidance of doubt, the terms “consists of”, “consisting of”, andrelated terms, are intended to be interpreted in an exclusive mannerThus, the PMCV VLP of the invention is composed of multiple copies ofPMCV ORF1 protein only. Specifically, the PMCV VLP does not include anyother proteins that are not, or cannot be considered to be, PMCV ORF1proteins, or indeed any other materials whatsoever. For example, thePMCV VLP does not contain any other PMCV proteins or nucleic acids, suchas PMCV ORF3 (or ORF2) protein, and the PMCV VLP does not contain anyexogenous proteins or nucleic acids, for example, derived from any partof the cell or expression system used in the production of the PMCV VLP.In addition, for the avoidance of doubt, the phrase “consists only ofPMCV ORF1 protein” is to be understood to mean that the virus-likeparticle consists of a plurality of PMCV ORF1 proteins only, and,specifically, the “consists of” language is to be understood to reflectthat the VLP is composed only of a plurality of PMCV ORF1 proteins anddoes not contain any other proteins or materials of any kind.

The PMCV VLP preferably consists of multiple copies of identical PMCVORF1 proteins. In some embodiments, however, any given two of the PMCVORF1 proteins of an individual PMCV VLP may differ. Thus, in someembodiments, the nucleic acid or protein sequences of any given two ofthe PMCV ORF1 proteins of an individual PMCV VLP may have at least 70%sequence identity. For example, the nucleic acid or protein sequences ofany given two of the PMCV ORF1 proteins of an individual PMCV VLP mayhave at least 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity.Preferably, any given two of the PMCV ORF1 proteins of an individualPMCV VLP may differ by no more than 15, 12, 10, 8, 7, 6, or 5 amino acidresidues. Even more preferably, any given two of the PMCV ORF1 proteinsof an individual PMCV VLP may differ by only 4, 3, 2, or 1 amino acid.

In preferred embodiments, the PMCV VLP of the invention are immunogenicin fish, and can be defined and identified on the basis of theirimmunogenic properties. The term “immunogenic” refers to the propertythat, when administered to fish (preferably salmonids), optionallytogether with an effective amount of a suitable adjuvant, in animmunogenically effective amount, the VLP of the invention induce theproduction of PMCV-specific antibodies in the fish. For the avoidance ofdoubt, an “effective amount” refers to an amount effective, at dosagesand for periods of time necessary under normal circumstances, to achievethe desired result.

Thus, in some embodiments, the PMCV VLP, when administered in animmunologically effective amount, optionally together with an effectiveamount of a suitable adjuvant, may be capable of inducing the productionin fish of neutralizing antibodies against PMCV.

In some embodiments, the PMCV VLP of the invention may be identified bythe ability to bind an antibody raised against the PMCV ORF1 protein asdefined in SEQ ID No: 2, such as a recombinant PMCV ORF1 protein made byany suitable method. Thus, the binding affinity between an antibodyraised against the PMCV ORF1 protein of SEQ ID No: 2 and the PMCV VLP ofthe invention may be at least 50% of the binding affinity between thesame antibody and the PMCV ORF1 protein of SEQ ID No: 2. Preferably, thebinding affinity of the antibody for the PMCV VLP of the invention is atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and may be 100%,of the binding affinity of the antibody for the PMCV ORF1 protein of SEQID No: 2. Methods for the production of suitable detection antibodiesare known. For example, such neutralizing antibodies can be polyclonal,such as those produced in immunized rabbits. Alternatively, antibodiesmay be polyclonal, for example produced as a hybridoma with B cells fromimmunized mice.

Such an antibody may be an antibody raised in fish such as salmonids.Thus, in some embodiments, the PMCV VLP may be capable of eliciting animmune response in fish vaccinated with recombinant PMCV ORF1 protein.

For the avoidance of doubt, the “binding affinity” refers to thestrength of the sum total of noncovalent interactions between a singlebinding site of a molecule (such as an antibody) and its bindingpartner. Unless otherwise stated, “binding affinity” in relation to thepresent invention refers to intrinsic binding affinity which reflects a1:1 interaction between members of a binding pair. The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (Kd). A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for the purposesof the present invention.

Medical Use

For the first time, PMCV VLP can be made. The PMCV VLP structurallymimic wild-type virus particles and, as a result, are immunologicallycomparable to inactivated whole viruses.

Thus, the PMCV VLP of the invention may be suitable for use, orformulated for use, in or as a medicament. For the avoidance of doubt, a“medicament” is an agent or composition that is active to treat orprevent the disorder in question or its symptoms, or side effects. Asused herein, “treatment” refers to clinical intervention in an attemptto alter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of disease.Desirable effects of treatment may, for example, include preventingoccurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and/or remission or improved prognosis.

In some embodiments the PMCV VLP is suitable for use in treating orpreventing viral infections of fish. In some embodiments the PMCV VLP issuitable for use in treating or preventing cardiomyopathy syndromeinfections of fish, including salmonid fish.

Vaccine

The PMCV VLP of the invention may be used in the production of avaccine. For the avoidance of doubt, the term “vaccine” refers to amaterial or composition that can produce an immune response that blocksthe infectivity, either partially or fully, of an infectious agent,which in respect of the present invention is the virus PMCV which causesCMS, affecting fish such as Atlantic salmon. Thus, when the vaccines ofthe invention, comprising PMCV VLP consisting only of PMCV ORF1 protein,are administered to a fish, the fish is immunised against CMS caused byPMCV. A vaccine comprising the PMCV VLP of the invention may beformulated using known techniques. Preferably vaccines comprising thePMCV VLP of the invention may also comprise an effective amount of asuitable adjuvant.

Thus, another embodiment relates to a vaccine for the treatment orprevention of CMS infection of salmonid fish. The vaccine comprises thePMCV VLP of the invention. The vaccine may further comprise an effectiveamount of a suitable adjuvant.

Generally, the PMCV VLP of the invention may be formulated and used in avaccine or other medicinal composition in ways and by means exactly thesame as those known to the skilled person in relation to otherimmunogenic virus particles, including attenuated or inactivatedviruses, and other VLPs.

For example, the vaccine may further comprise other additives that areused in the art for the production of such compositions, such as acarrier, a diluent, an adjuvant, a stabilizer, an emulsifier, or otheragent, as described below.

Carriers

In some embodiments, the vaccine further comprises a pharmaceuticallyacceptable carrier and/or diluent. Examples of pharmaceuticallyacceptable carriers or diluents useful in the present invention includesterile water, saline, aqueous buffers such as PBS and others, culturemedium, carbohydrates (such as sorbitol, mannitol, starch, sucrose,glucose, dextran), proteins (such as albumin or casein), proteincontaining agents such as bovine serum or skimmed milk, and buffers(such as phosphate buffer).

Adjuvants

In some embodiments, the vaccine further comprises an adjuvant.Adjuvants are non-specific stimulators of the immune system. Theyenhance the immune response of the host to the vaccine. Examples ofsuitable adjuvants known in the art include lipopolysaccharide, CpG,Freunds Complete and Incomplete adjuvant, vitamin E, non-ionic blockpolymers, muramyldipeptides, ISCOMs (immune stimulating complexes),Saponins, mineral oil, vegetable oil, and Carbopol. Oil adjuvantssuitable for use in water-in-oil emulsions include, for example, mineraloils or metabolisable oils. Mineral oils include, for example, Bayol®,Marcol®, and Drakeol®. An example of a non-mineral oil adjuvant isMontanide-ISA-763-A. Metabolisable oils include, for example, vegetableoils, such as peanut oil and soybean oil, animal oils such as the oilssqualane and squalene from shark liver or plants, and tocopherol and itsderivatives.

Stabilisers

In some embodiments, the vaccine further comprises a stabiliser. Astabilizer can be added to the vaccine, for example, to protect it fromdegradation, to enhance the shelf-life, or to improve freeze-dryingefficiency. Useful stabilizers include SPGA, skimmed milk, gelatine,bovine serum albumin, formalin, carbohydrates (such as sorbitol,mannitol, trehalose, starch, sucrose, dextran or glucose), proteins(such as albumin or casein or degradation products thereof), andbuffers, such as alkali metal phosphates.

Emulsifiers

In some embodiments, vaccines comprising the PMCV VLP of the inventionfurther comprise one or more suitable surface-active compounds oremulsifiers, such as Span®, Cremophore®, or Tween®.

Form of Vaccine

Vaccines comprising the PMCV VLP of the invention may be prepared in anysuitable form, including forms generally used for the production andpreparation of viral vaccines. For example, the vaccine of the inventionmay be in the form of an emulsion, a suspension, in a lyophilized formor, alternatively, in a frozen form. If the formulation is to be frozen,glycerol or other similar agents may be included in the formulation toenhance the stability of the VLP when frozen. Reconstitution isadvantageously effected in sterile water or a suitable buffer.

Fish

Vaccines comprising the PMCV VLP of the invention are preferablyadministered to fish, which may, for example, be any species of fishthat is susceptible to CMS infection. Of particular note, the vaccine issuitable for administration to, and for treating, a fin fish, which maybe a telostei, for example, of the order Salmoniformes. Preferably, theclaimed formulation may be used to treat salmon such as Atlantic andPacific salmon, such as Coho salmon, arctic char, and trout such asrainbow trout and brown trout.

Route of Administration

The present invention includes methods of vaccinating fish against CMSinfection. Methods comprise administering to a fish a vaccinecomposition comprising the PMCV VLP of the invention, optionallytogether with a suitable adjuvant.

The vaccine composition may be administered to the host in any suitablemanner known in the art. In particular, vaccine formulations comprisingthe PMCV VLP of the invention may be suitable for parenteraladministration, such as by intraperitoneal injection. Other conventionalfish vaccination methods may also be used if appropriate, includingimmersion, dipping, or oral administration.

Dosage

Vaccines comprising the PMCV VLP of the invention are preferably used,optionally together with a suitable adjuvant, inimmunologically-effective amounts. For the avoidance of doubt, in thiscontext, the expression “immunologically-effective amount” means theamount of PMCV VLP, optionally together with an adjuvant, that isrequired to stimulate the production of protective levels of immunity ina host. Thus, the PMCV VLP of the invention is used in an amount, or ina composition, that is sufficient to induce an immune response in fishthat decreases the pathological effects caused by infection with awild-type PMCV, in comparison to the pathological effects caused byinfection with a wild-type PMCV in non-immunized fish.

The dosage of the vaccine (and the route of administration) employedwill be dependent on various factors including the size and weight ofthe host, the vaccine formulation (such the presence of an adjuvant),and the timing of the administration. Generally, dosages may need to beincreased for larger, more robust fish, and decreased for smaller, moredelicate fish.

Vaccines comprising the PMCV VLP of the invention can generally beadministered by injection in a dosage of 10³ to 10¹² VLP, such as 10⁴ to10¹¹ VLP, 10⁵ to 10¹⁰ VLP, or 10⁶ to 10¹⁰ VLP. Preferably the dosagecontains in the range of 10⁷ to 10⁹ VLP more preferably between 10⁸ and10⁹ VLP per dose.

Combination Vaccines

In some embodiments, vaccines comprising the PMCV VLP of the inventionare combination vaccines and further comprise animmunologically-effective amount of at least one additional immunogenicagent. Thus, another embodiment relates to a vaccine for the treatmentor prevention of CMS infection and one or more other infections of fish.The vaccine comprises the PMCV VLP of the invention together with animmunogenic amount of at least one other antigen against afish-pathogenic microorganism or a fish-pathogenic virus.

The at least one other vaccine may comprise an antigen from a bacterialsource, a viral source, a parasitical source, and/or a fungal source.Polyvalent vaccines containing antigens from typical fish pathogens arewell known in the art and are already commercially available.

The antigen from a bacterial source may be selected from the groupcomprising: live, attenuated or killed bacteria of the speciesPiscirickettsias sp. Aeromonas sp., Vibrio sp., Listonella sp.,Moritella viscosa, Photobacterium damsela, Flavobacterium sp., Yersiniasp., Renibacterium sp., Streptococcus sp., Lactococcus sp., Leuconostocsp., Bifidobacterium sp., Pediococcus sp., Brevibacterium sp.,Edwarsiella sp., Francisella sp., Pseudomonas sp., Cytophaga sp.,Nocardia sp., and Mycobacerium sp.

The antigen from a viral source may be selected from the groupcomprising:

Viral Hemorrhagic Septicemia Virus (VHSV), Infectious HematopoieticNecrosis virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV),Spring Viremia of Carp (SVC), Channel Catfish Virus (CCV), InfectiousSalmon Anaemia virus (ISAV), pancreatic disease virus (SPDV),Iridovirus, and piscine orthoreovirus (PRV).

The antigen from a parasitic source may be selected from the groupcomprising: Lepeophtheirus Sp., Caligus Sp., and Ichthyophthirius Sp.

The antigen from a parasitic source may be selected from the groupcomprising: Saprolegnia Sp., Branchiomyces sanguinis, Branchiomycesdemigrans and Icthyophonus hoferi.

Method of Manufacture

The PMCV VLP of the invention may be manufactured by any suitablemethod. In preferred embodiments the PMCV VLP of the invention are madein a baculovirus based expression system.

Baculovirus expression systems used in conjunction with insect cellshave become well-established for the production of proteins. In suchsystems, a recombinant baculoviral vector is used to introduce the geneof interest (in this case PMCV ORF1) into insect cells under the controlof a strong baculoviral promoter. Infection of the insect cells in thisway results in replication of the recombinant baculovirus vector genome,thereby increasing the number of genetic templates that encode the geneof interest and increasing the level of recombinant protein expression.Baculovirus-mediated protein expression also provides correct folding ofrecombinant proteins and other important post-translationalmodifications that provide proper biological activity and function tothe expressed proteins. The insect cells used in the system can be grownon serum free media which may have further advantages in terms of costsand biosafety.

Thus, in some embodiments, the invention relates to a baculovirusexpression vector comprising the PMCV ORF1 gene, which is defined forthe purposes of the present invention as all of the genetic informationencoding the expression of a PMCV ORF1 protein. In preferred embodimentsthe genetic information encodes the expression of a PMCV ORF1 proteinonly. Thus, the invention also relates to a baculovirus expressionvector comprising a polynucleotide encoding at least PMCV ORF1 protein,such as PMCV ORF1 protein only.

For example, the invention also relates to a baculovirus expressionvector comprising the nucleotide sequence of SEQ ID No: 1. In someembodiments, the invention relates to a baculovirus expression vectorcomprising a nucleotide sequence encoding a PMCV ORF1 protein asdescribed above, which is capable of forming a PMCV VLP.

The invention also relates to the use of a baculovirus based expressionsystem for the production of PMCV VLP consisting only of PMCV ORF1protein.

The invention also relates to a method for the production of PMCV VLP ina baculovirus based expression system, comprising the steps of:

-   -   a) infecting an insect cell line with a baculovirus expression        vector encoding the expression of PMCV ORF1 protein; and,    -   b) suitably incubating the infected cells to allow the        expression of the PMCV ORF1 protein and the production of the        PMCV VLPs.

The invention also relates to a method for the production of animmunogenic composition comprising PMCV VLP. Optionally the methodcomprises steps a) and b) above and further comprises the step ofpreparing an immunogenic composition comprising the PMCV VLP.

Cells

Any suitable cell line may be used for the production of the PMCV VLP.The cells preferably used for the expression of PMCV ORF1 and productionof the VLPs are cells that are suitable for use in a baculovirusexpression system. Preferred cell lines include Sf21 cells (originallyderived from the pupal ovarian cells of Spodoptera frugiperda), Sf9cells (which are a clonal isolate of Sf21), and Tni (also called T. nior TnHi5™) cells (which are originally derived from the ovarian cells ofTrichoplusia ni (cabbage looper).

PMCV ORF1 protein has surprisingly been found to be expressed insignificantly greater quantities in Tni cells than in other cell types,and thus Tni are the preferred cell type for use in the invention.

Baculovirus Expression System

There are a number of commercial systems available for expressingrecombinant proteins using baculovirus, including flashBAC™ (OxfordExpression Technologies EP 1 144 666), BackPack™ (BD BiosciencesClontech), BacVector® 1000/2000/3000 (Novagen®). BAC-TO-BAC®(Invitrogen™), and BaculoDirect™ (Invitrogen™). All of these systems arebased on the principle of expressing recombinant proteins by placingthem under the control of the very late baculovirus promoters polh orp10. In preferred embodiments of the invention the PMCV VLP are producedusing the flashBAC™ system.

Infection

Cells to be infected with the baculovirus expression vector encoding theexpression of PMCV ORF1 protein may be cultured at a density of about1×10⁴-1×10⁸ cells/ml, such as 1×10⁵-1×10⁷ cells/ml, and preferably thecells are cultured at a density of about 1×10⁶ cells/ml.

The vector is added to the cells at a multiplicity of infection (MOI) ofabout 0.5-5, such as about 1-3, or preferably about 2. For the avoidanceof doubt, the MOI is defined for the purposes of the present inventionas the ratio of agents (in this case baculovirus expression vectors) toinfection targets (in this case cells of a suitable insect cell line).Following addition of the vector to the cell culture, the cells arepreferably incubated at a temperature of 25-29° C., such as at or about26° C., 27° C., or 28° C. The cells are preferably incubated at thistemperature for a period of about 12-36 hours, such as about 18, 21, 24,27, or 30 hours.

Incubation

Incubating the infected cells to allow the expression of the PMCV ORF1protein and the production of the PMCV VLPs generally involvesincubating the cells for at least 1, 2, or 3 days and up to about 4, 5,6, 8, or 10 days. Preferably, the cells are incubated for about 1-6days, and most preferably about 2-4 days.

Baculovirus expression systems are always used at or close to 26-28° C.because that is the optimal temperature for both the infection processand the expression process. The use of this temperature is consistentwith both the manufacturer's instructions, and the fact that the systemwould be expect to optimised to the host body temperature.

However, as detailed in the following Examples, it has surprisingly beenfound that if this temperature is used to incubate the cells infectedwith a baculovirus expression vector comprising PMCV ORF1, the proteinis produced in significant quantities but does not form PMCV VLP.

In IMNV and other totiviruses that are capable of infecting new hostcells, the proteins are translated as a precursor protein which is thenproteolytically cleaved. However, PMCV ORF1 is not processed in any way,and in particular, does not require proteolytic cleavage or any otherpost-translational modification in order to form the mature PMCV ORF1protein. It was, therefore, extremely unexpected to observe productionof the PMCV ORF1 protein but no VLP. It has now surprisingly been found,however, that PMCV VLP can be produced if the incubation temperatureused to allow the expression of the PMCV ORF1 protein and the productionof the PMCV VLP is significantly below the temperature prescribed by themanufacturers of the expression system and used conventionally in theart.

Thus, in preferred embodiments, the incubation step of the methodcomprises incubating the cells at a temperature in the range of 12-20°C., such as 14-18° C., and preferred incubation temperatures are 15, 16,or 17° C.

Preparing a PMCV VLP Composition

Optionally the method for the production of PMCV VLP in a baculovirusbased expression system includes a final step of isolating the PMCV VLPsfrom the culture medium. Suitable methods of isolating/purifying the VLPfrom the cell culture will be known to the skilled person and anysuitable method may be used.

The isolation may be comprehensive, to provide essentially purified PMCVVLP, or may be less comprehensive, for example, involving simply theremoval of cell debris and remaining baculoviruses such as by a suitablefiltration method.

In some embodiments, the VLP are not isolated to any extent from thecell culture prior to the production of a vaccine or other medicinalcomposition. In such embodiments, further non-isolating treatment of thecell culture may nevertheless be used, such as formalin or heattreatment of the cultures or a treatment to lyse the cells.

EXAMPLES

The invention will now be explained in further detail in the followingExamples, which demonstrate the development of the claimed PMCV VLP,consisting only of PMCV ORF1 protein.

Example 1. Production of Recombinant Baculoviruses

Recombinant baculoviruses were constructed using the flashBAC systemfrom Oxford Expression Technologies following the manufacturer'sinstructions. PMCV ORF1 and PMCV ORF3 genes with an added His-tag werecloned into the transfer vector.

Recombinant baculoviruses were produced by co-transfection of Sf9 insectcells with duplicate reaction mixtures, each containing flashBACUltraviral DNA (100 ng) and transfer vector DNA (500 ng) for ORF1 or ORF3,together with Lipofectin liposome forming reagent (Invitrogen).

The mix was added to 35 mm² dishes with Sf9 insect cells seeded at adensity of 1×10⁶ cells/dish. The dishes were then incubated at 28° C.for 5 days, following which the medium containing each virus washarvested into sterile tubes.

Stocks of the recombinant PMCV ORF1 and PMCV ORF3 baculoviruses werethen made, and titres determined, following the manufacturer'sinstructions.

Example 2. Protein Expression

Sf9 cells were seeded in 35 mm² dishes at 1×10⁶ cells/dish, and Tnicells were seeded at 0.5×10⁶ cells/dish. Each dish was infected with thevirus (MOI=5).

Following incubation for 72 hrs at 28° C. the cell pellets andsupernatants were harvested from each dish and run on a pre-cast 4-16%NuSep LongLife SDS-PAGE gel (Generon) with Fermentas PageRulerPrestained protein ladder Plus. Negative control was also included.Western blots were prepared using anti-His antibody at a dilution of1:1000. Visualisation was by anti-mouse antibody: alkaline phosphataseconjugate (1:30,000 dilution) using detection reagents NBT and BCIP. Thesize (mass) of the ORF1 protein was approximately 100 kDa, and the size(mass) of the ORF3 protein was slightly greater than 35 kDa.

Both Sf9 and Tni cells were found to express both ORF1 and ORF3 protein,but Tni cells expressed the proteins in much greater quantities than Sf9cells.

Example 3. Protein Expression

Expression experiments were carried out by infecting 20 ml cultures ofTni cells (at 1×10⁶ cells/ml) at MOI=2 with the ORF1, ORF3, or acombination of the ORF1 and ORF3, recombinant baculoviruses.

The cells were incubated at 28° C. until 96 hours post-infection (hpi).The cell pellets were spun down at 4,000 rpm for 15 minutes and thepellet resuspended in 10 ml PBS. The resuspended pellets were lysed byfreeze thawing, and the samples were centrifuged at 4,000 rpm for 30minutes to remove cell debris. The supernatants were analysed by Westernblot. The results are shown in Table 1, in which “+” indicates that aprotein band was observed on the Western blot, and “-” indicates that noprotein band was observed.

TABLE 1 Expressed Detection on Western blot Protein(s) ORF1 (100 kDa)ORF3 (35 kDa) ORF1 + − ORF3 − + ORF 1 + ORF3 + +

Results: This experiment shows that both ORF1 and ORF3 were expressedwell in Tni cells using a baculovirus system.

Example 4. Detection of VLPs

Particles of the size expected for PMCV VLPs are pelleted bycentrifugation at ˜100,000 g. Thus, any PMCV proteins contained withinvirus-like particles will be observed on the Western blot in the pelletfraction produced under these centrifugation conditions.

Supernatants produced as described in Example 3 were centrifuged at˜100,000×g for 30 minutes to pellet any virus like particles. Theresulting supernatants and pellets were analysed by Western blot.

The results are shown in Table 2, in which “+” indicates that a proteinband was observed on the Western blot, and “-” indicates that no proteinband was observed.

TABLE 2 Band detected on Western blot Fraction Supernatant PelletProtein(s) 100 kd 35 kD 100 kD 35 kD expressed (ORF1) (ORF3) (ORF1)(ORF3) ORF1 + − − − ORF3 − + − − ORF 1 + ORF3 + + − −

Results: Both ORF1 and ORF3 proteins were expressed in significantlevels as soluble proteins in the cell culture and detected insignificant quantities in the supernatant fraction. However, neitherORF1 nor ORF3 proteins were detected in the pellet fraction. Thus,although ORF1 and ORF3 proteins were observed to be expressed insignificant quantities, no virus-like particles were formed. This wasthe case when ORF1 and ORF3 proteins were expressed individually or incombination.

Example 5. Protein Expression and Detection of VLP

The ORF1 and ORF3 proteins were found to be expressed efficiently underthe incubation conditions conventionally used and in accordance with themanufacturer's instructions, but despite the significant expression ofthe ORF1 and ORF3 proteins, no PMCV VLP were observed to be formed.

To determine whether the expressed PMCV ORF1 and/or ORF3 proteins couldbe induced to form VLP, the protein expression experiments were repeatedin which different variables of the incubation conditions wereinvestigated, including the MOI, the incubation time, and the incubationtemperature.

The expression experiments were carried out by infecting 20 ml culturesof Tni cells (1×10⁶ cells/ml) at MOI=2 with the ORF1, ORF3, or acombination of the ORF1 and ORF3, recombinant baculoviruses.

Adjusting different experimental parameters and conditions, includingthe MOI and the incubation time, did not result in the production ofsignificant quantities of PMCV VLP.

To investigate the effect of incubation temperature on the production ofPMCV VLP, cells were infected by incubation at 28° C. for 24 hrs beforebeing incubated at 15° C. until 120 hpi. The cells were harvested andpelleted by centrifugation at 4,000 rpm for 15 minutes and the washedpellets were resuspended in 10 ml PBS. The resuspended pellets werelysed by freeze thawing, and the samples were centrifuged at 4,000 rpmfor 30 minutes to remove cell debris. The supernatants were collectedand centrifuged at ˜100,000 g for 30 minutes to pellet any virus likeparticles.

The resulting pellets were resuspended in 0.5 ml PBS and used for bothwestern blot and electron microscopy analysis (see below). Thesupernatants were also analysed by Western blot (described in Example6). The results are shown in Table 3, in which “+” indicates that aprotein band was observed on the Western blot, and “-” indicates that noprotein band was observed.

TABLE 3 Detection on Western blot Fraction Supernatant Pellet ExpressedORF1 ORF3 ORF1 ORF3 Protein(s) (100 kDa) (35 kDa) (100 kDa) (35 kDa)ORF1 − − + − ORF3 − + − − ORF 1 + ORF3 − + + −

Results: When PMCV ORF3 protein only was expressed, by incubating theinfected cells at 15° C., no virus like particles were found to beformed. After centrifugation of the cell lysates at ˜100,000 g for 30minutes ORF3 protein was detected in the supernatant fraction only andnot in the pellet fraction.

When both PMCV ORF1 and ORF3 proteins were expressed simultaneously, byincubating the infected cells at 15° C., virus-like particles wereformed, but surprisingly the virus-like particles were found by Westernblot to be composed solely of ORF1 protein, and in particular, ORF3protein was not detected in the pellet fraction by Western blot. Thus,although ORF1 and ORF3 proteins were expressed in significant levels inthe cells, the PMCV VLP produced contained ORF1 protein only and no ORF3protein.

When PMCV ORF1 protein only was expressed, by incubating the infectedcells at 15° C., virus like particles were found to be produced. Thevirus like particles were found by Western blot to contain ORF1 protein.Moreover, the formation of the virus like particles consisting only ofPMCV ORF1 protein was found to be highly efficient because very littleORF1 protein was detected in the supernatant fraction. Thus,substantially all of the ORF1 protein that was expressed in the cellswas found to be incorporated into virus like particles.

Each of the pellet fractions produced by centrifuging the cell lysatesat 100,000 g for 30 minutes was also analysed by electron microscopy(see Example 6).

Example 6. Electron Microscopy

The re-suspended pellet fractions described in Example 5 were processedfor electron microscopy. Samples were applied to Formvar andcarbon-coated grids and adsorbed alone for 5 minutes and then with fivedroplets of 2% ammonium molybdate. Excess liquid was wicked away and thesamples were air dried. The grids were examined with a Hitachi H7650transmission electron microscope.

An example of one of the images obtained by electron microscopy of thepellet fraction of a sample in which PMCV ORF1 protein only wasexpressed at 15° C., as described above (in Example 5), is shown inFIG. 1. FIG. 2 shows an example image obtained by electron microscopy ofthe sample produced by the expression of ORF1 and ORF3 proteins incombination.

The electron microscopy images of FIGS. 1 and 2 show round structureswith a diameter of about 30-40 nanometres. These structures are exactlyas expected for empty PMCV particles both in predicted shape and size.

No virus-like particles were observed in the pellet fraction of samplesin which PMCV ORF3 protein only was expressed.

Example 7. Vaccine Containing PMCV VLP Antigen

Antigen was produced by infecting 20 ml cultures of Tni cells (1×10⁶cells/ml) at MOI=2 with the ORF1, ORF3, or a combination of the ORF1 andORF3, recombinant baculoviruses.

The cells were incubated at 28° C. for 24 hrs before being placed at 15°C. until 120 hpi, as described above in Example 4. The Tni cells werespun down at 4,000 rpm for 15 minutes and the cell pellets werere-suspended in 10 ml PBS. The re-suspended pellets were lysed by freezethawing, and samples were centrifuged at 4,000 rpm for 30 minutes toremove cell debris. The supernatants were harvested, and the amount ofVLP quantified by Western blot. Remaining baculoviruses were inactivatedby the addition of formalin.

Vaccines were formulated as a water-in-oil emulsion using standardtechniques.

Example 8. Vaccination and Challenge Trial

Atlantic salmon post smolts (weighing about 30 g each) were injectedwith vaccine comprising PMCV VLP prepared as described above (in Example7).

Six weeks post vaccination the fish were challenged intramuscularly witha preparation comprising homogenised heart tissue from fish diagnosedwith severe CMS.

At 7 weeks post challenge, changes in the ventricle of the hearts of thevaccinated fish was determined by histological analyses using standardmethods.

Results:

Score 1 Score 2 Score 3 VLP vaccine 100% — — Control vaccine (with  60%40% — irrelevant antigens)

The results show that no fish vaccinated with VLP vaccine had severepathology in the heart ventricle, while 40% of the fish vaccinated witha control vaccine had severe pathology in the heart ventricle. Thisshows that a vaccine using ORF1 VLP as antigen can protect salmonagainst severe pathology in the heart ventricle.

In order to address various issues and advance the art, the entirety ofthis disclosure shows by way of illustration various embodiments inwhich the claimed invention may be practiced and provide an PMCV VLPconsisting only of PMCV ORF1 protein. The advantages and features of thedisclosure are of a representative sample of embodiments only, and arenot exhaustive and/or exclusive. They are presented only to assist inunderstanding and teach the claimed features. It is to be understoodthat advantages, embodiments, examples, functions, features, and/orother aspects of the disclosure are not to be considered limitations onthe disclosure as defined by the claims or limitations on equivalents tothe claims, and that other embodiments may be utilized and modificationsmay be made without departing from the scope and/or spirit of thedisclosure. Various embodiments may suitably comprise, consist of, orconsist essentially of, various combinations of the disclosed elements,components, features, parts, steps, means, etc. In addition, thedisclosure includes other inventions not presently claimed, but whichmay be claimed in future.

The invention claimed is:
 1. A recombinant piscine myocarditis virus(PMCV) virus-like particle (VLP) comprising a PMCV ORF1 protein, havingat least about 90% sequence identity to the sequence comprising SEQ IDNo:
 2. 2. The recombinant PMCV VLP of claim 1, comprising the PMCV ORF1protein having at least about 95% sequence identity to the sequencecomprising SEQ ID No:
 2. 3. The recombinant PMCV VLP of claim 2,comprising the PMCV ORF1 protein having at least about 99% sequenceidentity to the sequence comprising SEQ ID No:
 2. 4. The recombinantPMCV VLP of claim 1, comprising the PMCV ORF1 protein having a sequencediffering from the PMCV ORF1 protein sequence comprising SEQ ID No: 2 byno more than 80 amino acid residues.
 5. The recombinant PMCV VLP ofclaim 4, comprising the PMCV ORF1 protein having a sequence differingfrom the PMCV ORF1 protein sequence of SEQ ID No: 2 by no more than 15amino acid residues.
 6. A method for the production of a recombinantPMCV VLP comprising the PMCV ORF1 protein of claim 1, the methodcomprising: a) infecting an insect cell with a baculovirus expressionvector encoding the recombinant PMCV ORF1 protein of claim 2; and, b)incubating the infected cell of step (a) at a temperature in the rangeof 12-20° C. to allow the expression of the PMCV ORF1 protein in orderto produce a PMCV VLP.
 7. The method as in claim 6, wherein theincubation of the infected cell comprises incubating the cell at atemperature for a period of 1-10 days.
 8. The method as in claim 7,wherein the incubation of the infected cell comprises incubating thecell at a temperature in the range of 15-17° C.
 9. A PMCV VLP obtainedby the method of claim
 6. 10. A method of prevention of cardiomyopathysyndrome (CMS) infections in fish, the method comprising administeringto the fish in need thereof the PMCV VLP according to claim
 1. 11. Avaccine comprising the PMCV VLP of claim
 10. 12. The vaccine of claim 11further comprising at least one other antigen of a fish-pathogenicmicroorganism or a fish-pathogenic virus.
 13. A baculovirus expressionvector comprising the PMCV ORF1 protein as defined in claim
 1. 14. Aninsect cell comprising a baculovirus expression vector of claim 13.